Proteins and peptides serve multifunctional roles in biological systems. As ligands for various receptors as well as substrates for various enzymes, peptides can be used to regulate biological processes wherein the function of proteins and peptides can be defined by the structure, orientation and positioning of side-chains in aqueous solution as determined by secondary and tertiary structure. However, the primary sequence of amino acid residues needed for proper orientation in aqueous solution can sometimes lead to instability. Due to the difficulties in delivering peptide-based drugs, relatively few peptide-based drugs are on the market. Although insulin and insulin derivatives are one of the first commercially formulated peptide based drugs, insulin has several structural features allowing for insulin to remain stable during long storage periods in solution compared with small peptides and particularly compared with small peptides synthesized through solid-phase synthesis rather than recombinant methods expressed inside a living cell. Without being limited to any one theory, human insulin, including human insulin recombinantly expressed in bacterial cells, is formed of two separate peptide chains having 21 and 30 amino acid residues, respectively. The two peptide chains are linked through three disulfide bridges between pairs of cysteine residues. Both peptide chains form a significant amount of alpha-helical secondary structure. Due to the disulfide bridges linking the peptide chains, the alpha-helical regions of the two peptide chains contact one another forming numerous salt bridges and van der Waals contacts. As a result, insulin has a well-ordered tertiary structure that stabilizes insulin against surface adsorption by reducing the exposure of hydrophobic regions to a surrounding aqueous environment. Further, the structure of insulin reduces mobility of the peptide backbone helping to protect insulin from proteolytic attack from acids or bases. Insulin in solution can form hexamers mediated by zinc ions, which further stabilize its structures. Many commercial formulations of insulin contain zinc salts to promote stability.
Natriuretic peptides have a structure allowing for the binding to atrial natriuretic peptide (ANP) receptor, which controls the activity of an associated guanylyl cyclase. The binding of an agonist ligand to the ANP receptor results in several physiological effects including decrease in cardiac volume and blood output, decrease in blood pressure and increase in glomerular filtration rate (GFR). Without being limited to any single theory, the natriuretic peptides are not believed to have significant amounts of secondary structure, such as alpha-helices. A lack of well-ordered secondary structure may possibly allow for a high degree of freedom of movement in the peptide chain, which can open the peptide chain to attack by proteolytic enzymes and acid/base attack as well as other chemical reactions such as deamidation. Hence, certain peptides and polypeptides, such as natriuretic peptides, may be rapidly degraded when formulated into a solution for administration. In particular, the amide bonds forming the peptide backbone can be subject to nucleophilic attack and hydrolysis in aqueous solutions. Further, peptides can be degraded by peptidases, amidases, and/or esterases present in the environment.
Stable formulations of therapeutic agents are particularly important for use in delivery devices that expose peptides to elevated temperatures, mechanical stress and/or hydrophobic interactions with components of delivery devices. Formulations of peptides should remain soluble and substantially free of aggregation, even though subjected to the subject's body heat and motion for periods ranging from a few days to several months. Of the 20 amino acids that form most natural peptide sequences, many have side chains that are hydrophobic, where peptides containing a high amount of such hydrophobic amino acid residues may have limited solubility in aqueous solution or undergo aggregation over time. For this reason, some peptides may have limited therapeutic use. Even in situations where a peptide has pharmacological effect when administered, the concentration of the peptide in an aqueous pharmaceutical composition can be unstable. Depending on the particular administration requirements and time limitations, a formulation with a short shelf-life may have little practical value. While organic solvents increase the solubility of most peptides, the presence of organic solvents in compositions for injection can be problematic. Chemical modifications of peptides to increase solubility are also known. Such chemical modifications can take the form of substitution of specific amino acid residues as well as covalent attachment to the N- and/or C-terminus of groups serving to increase solubility. However, without being limited to any particular theory, chemical modification can undesirably decrease the biological efficacy of the peptide. Hence, there is a need for a stable formulation of one or more chimeric natriuretic peptides having a long-shelf life that can be stably used in mechanical delivery or implantable devices for protracted periods of time. There is also a need for a method for preparing such stable chimeric natriuretic peptide formulations.